Although new therapies have had an impact prolonging the quality and quantity of life of men suffering from prostate cancer (PCa), novel treatments are urgently needed to combat aggressive forms of the disease. Recently, we made several key discoveries in the development of therapeutic peptides targeting the membrane bound metalloprotease aminopeptidase N (APN) in aggressive subtypes of PCa. To accurately and reliably interrogate the expression of APN in PCa tissues, we identified a novel antibody by phage display that specifically recognized APN in formalin-fixed paraffin embedded tissues. This antibody allowed us to screen PCa tissue microarrays (TMAs) with a fidelity never before seen. Immunohistochemistry analysis of patient biopsies and TMAs found that APN was preferentially overexpressed in non-androgen receptor (AR) driven PCa compared to AR-driven prostate adenocarcinoma. We also documented in a race disparity TMA and patient biopsies that APN was expressed in the PCa of African-American men while absent in Caucasian patients. Both African- American men and men with non-AR driven PCa represent patient populations who exhibit aggressive disease with poor overall survival. Using a rational design approach based off of the substrate specificity of APN, we identified a cyclic peptide (Leu-His-Ser-Pro-Trp = cLHSPW) that was a micromolar inhibitor of APN?s enzymatic activity. This peptide was found to be moderately therapeutic in APN-expressing PCa xenografts at high doses. Tethering of two cLHSPW peptides together by a linker created a dimer with enhanced potency in vitro and in vivo. The dimer was a nanomolar inhibitor of APN and was rapidly internalized by the cancer cell. When administered at the same high dose as the monomer, the dimer led to complete tumor attrition in vivo. These findings suggest that higher order multivalent cLHSPW peptides may be effective APN-targeted therapeutics. In this proposal, we will synthesize dimeric, trimeric, and tetrameric cLHSPW peptides using macrocyclic frames. In addition to ease of synthesis, the macrocyclic frames can also chelate radiometals for positron-emission tomography (PET). To develop a new class of PCa therapeutics, the in vitro therapeutic efficacy of the peptides will be characterized and PET imaging will be performed in vivo to monitor biodistribution (Aim 1), the ability of the peptides to eliminate tumor burden in subcutaneous xenografts will be examined (Aim 2) and X-ray crystallography and cryo-electron microscopy will be performed on APN complexed with the peptides to obtain mechanistic insights and ultimately lead to the development of optimized inhibitors (Aim 3). Our preliminary data strongly suggest that we have developed potent therapeutics that have the potential to result in a dramatic shift in how aggressive PCa is treated.